The genomes of eukaryotic organisms are highly organised within the nucleus of the cell. The long strands of duplex DNA are wrapped around an octomer of histone proteins (most usually comprising two copies of histones H2A, H2B H3 and H4) to form a nucleosome. This basic unit is then further compressed by the aggregation and folding of nucleosomes to form a highly condensed chromatin structure. A range of different states of condensation are possible, and the tightness of this structure varies during the cell cycle, being most compact during the process of cell division. Chromatin structure plays a critical role in regulating gene transcription, which cannot occur efficiently from highly condensed chromatin. The chromatin structure is controlled by a series of post translational modifications to histone proteins, notably histones H3 and H4, and most commonly within the histone tails which extend beyond the core nucleosome structure. These modifications include acetylation, methylation, phosphorylation, ubiquitinylation, SUMOylation. These epigenetic marks are written and erased by specific enzymes, which place the tags on specific residues within the histone tail, thereby forming an epigenetic code, which is then interpreted by the cell to allow gene specific regulation of chromatin structure and thereby transcription.
Histone acetylation is most usually associated with the activation of gene transcription, as the modification loosens the interaction of the DNA and the histone octomer by changing the electrostatics. In addition to this physical change, specific proteins bind to acetylated lysine residues within histones to read the epigenetic code. Bromodomains are small (˜110 amino acid) distinct domains within proteins that bind to acetylated lysine residues commonly but not exclusively in the context of histones. There are a family of around 50 proteins known to contain bromodomains, and they have a range of functions within the cell.
The BET family of bromodomain containing proteins comprises 4 proteins (BRD-2, BRD-3, BRD-4 and BRD-t) which contain tandem bromodomains (BD1 and 2) capable of binding to two acetylated lysine residues in close proximity, increasing the specificity of the interaction. BRD-2 and BRD-3 are reported to associate with histones along actively transcribed genes and may be involved in facilitating transcriptional elongation (Leroy et al, Mol. Cell. 2008 30(1):51-60), while BRD-4 appears to be involved in the recruitment of the pTEF-B complex to inducible genes, resulting in phosphorylation of RNA polymerase and increased transcriptional output (Hargreaves et al, Cell, 2009 138(1): 129-145). It has also been reported that BRD4 or BRD3 may fuse with NUT (nuclear protein in testis) forming novel fusion oncogenes, BRD4-NUT or BRD3-NUT, in a highly malignant form of epithelial neoplasia (French et al. Cancer Research, 2003, 63, 304-307 and French et al. Journal of Clinical Oncology, 2004, 22 (20), 4135-4139). Data suggests that BRD-NUT fusion proteins contribute to carcinogensesis (Oncogene, 2008, 27, 2237-2242). BRD-t is uniquely expressed in the testes and ovary. All family members have been reported to have some function in controlling or executing aspects of the cell cycle, and have been shown to remain in complex with chromosomes during cell division—suggesting a role in the maintenance of epigenetic memory. In addition some viruses make use of these proteins to tether their genomes to the host cell chromatin, as part of the process of viral replication (You et al Cell, 2004 117(3):349-60).
Umehara et al have solved the X-ray crystal structure for human BRD-2 BD1 when bound to a histone acetylated lysine residue (Protein crystallographic databank entry 2dvq) and demonstrated that the acetylated lysine residue accepts a hydrogen bond from the sidechain NH2 group of ASN 156 and also accepts a hydrogen bond from a water molecule that is itself hydrogen-bonded to the sidechain hydroxyl of TYR113. They have also predicted the amino acid residues which define the acetyl lysine recognition pocket of the first bromodomain (BD1) of human BRD-2 (JP2008-156311, The Institute of Physical and Chemical Research (RIKEN)). We have now identified small molecules which inhibit the binding of BD1 and 2 of the human BET family proteins BRD-2 to 4 to acetylated lysine residues of their physiological partner proteins. X-ray crystal studies of these molecules when bound to these BET bromodomains have allowed us to retrospectively identify the key binding sites involved in this interaction. This information can be used in the rational drug design of further small molecules which are able to inhibit the binding of the first and/or second bromodomains of human BRD-2 to 4 to acetylated lysine residues of their physiological partner proteins.